Heteropolymeric protein production methods

ABSTRACT

Cultured mammalian cells transfected with new vectors comprising full-length or partial α and β subunit genomic DNA sequences produce significantly higher levels of dimeric glycoprotein hormone than do cells transfected with α and β subunit cDNA sequences. In cases where only the cDNA clones are available, the cDNA sequences can be used in new expression vectors comprising introns or other important genomic regions from a homologous or heterologous source.

This is a continuation of copending application Ser. No. 07/368,628files on Jun. 20, 1989, now abandoned.

FIELD OF INVENTION

This invention relates to expression systems for the production ofheteropolymeric proteins from transformed mammalian cells and moreparticularly concerns novel expression systems and vectors for theproduction of dimeric glycoprotein hormones.

BACKGROUND OF THE INVENTION

Transfection of the α and β subunit cDNA clones into cultured mammaliancells has characteristically resulted in low gonadotropin expressionlevels. This has seriously impeded the production of these hormones on acommercial scale.

It is one aspect of the present invention to provide commerciallypractical methods for the production of such hormones.

While some genes, such as β-globin (1, 2) and immunoglobulin genes(3-5), require introns for optimal mRNA production, other genes, such asthymidine kinase (6), do not. Intron-dependent increases in geneexpression can result from either non-transcriptional (e.g. globingenes) or transcriptional (e.g. immunoglobulin genes) mechanisms.

Isolation of the genes which encode the human and bovine common α, FSHβ,LHβ, and human TSHβ subunits has been reported (8-14). Ramabhadran etal. (15) first described transfection with and subsequent expression ofthe human alpha subunit cDNA in mouse cells. Several groups have sincereported successful expression of dimeric glycoprotein hormones bytransfection of cultured mammalian cells. Some of these groups (16, 17)employed cDNA clones while others (14, 18, 19) have usedintron-containing cDNA or genomic sequences.

U.S. Pat. No. 4,840,866 and U.S. Pat. No. 4,923,805 describe the use ofcDNA clones (without introns) to produce gonadotropins in culturedmammalian cells. While those expression systems yield biologicallyactive molecules, the yield of the transformed mammalian cells aregenerally lower than described.

It is another aspect of the present invention to provide improvedexpression systems, useful with gonadotropins, which result in higheryield.

Matzuk and Boime (18a) mention that an intron inserted into the codingregion of the human α subunit cDNA improved expression results comparedwith the use of cDNA clones but provided no data to support thatcontention or specific description of their methods. In a recentpublication, Kaetzel and Nilson (7) reported relatively high levels ofbovine LH expression in CHO cells. Their system employed genomicsequences for expression of both the α and LHβ subunits. However, theeffect of genomic sequences versus cDNA sequences upon LH expression wasnot addressed in their paper.

It is yet another aspect of the present invention to obviate theconfusion represented by the present state of the art and to provide thecritical teaching necessary to derive improved vectors encoding dimericglycoproteins and production methods utilizing such vectors.

Introns have been linked to increased mRNA accumulation in tissueculture cells for rabbit β-globin (1,2,20), E. coli gpt (20), and mouseDHFR (20, 21). Examples of genes containing introns with enhancerelements which increase transcription are the immunoglobulin genes(3-5), the rat cytochrome c gene (22), and the human pro-α1(I) collagengene (23). Introns have also been shown to result in increasedtranscriptional efficiency in transgenic mice for the following genes:rat growth hormone, mouse metallothionein-I, and human β-globin (24).However, introns have no effect on the expression of these last threegenes when they are transfected into cultured mammalian cells.

It has been shown that expression levels can be influenced by different3' untranslated and polyadenylation regions (24, 25). For example,higher expression levels of a galK marker gene result if the bovinegrowth hormone polyadenylation region is used for transcriptiontermination rather than the SV40 early or human collagen polyadenylationregions (24).

SUMMARY

In accordance with the various principles and aspects of the presentinvention there are provided novel expression systems employing the useof α subunit genomic sequences, or α subunit cDNA constructions with anadded intron, which significantly and surprisingly enhance dimericglycoprotein hormone production in mammalian cells. This discovery willfacilitate the development of processes for high-yield production ofdimeric glycoprotein hormones which share the common α subunit. Theseinclude chorionic gonadotropin (GG), follicle stimulating hormone (FSH),luteinizing hormone (LH), and thyroid stimulating hormone (TSH).

It has also been unexpectedly discovered that TSHβ subunit geneexpression is intron-dependent. Characterization of the genomic regionsnecessary for optimal expression of the TSHβ subunit now made possibleby the instant invention provide critical and specific informationregarding the development of a process for efficient production ofdimeric TSH.

BRIEF DESCRIPTION OF THE DRAWINGS

Further understanding of the various aspects and principles of thepresent invention may be had by study of the accompanying figureswherein:

FIG. 1A and 1B: shows the strategy used to engineer the human α gene forexpression in tissue culture cells. Important restriction endonucleasesites are indicated. Filled-in boxes represent α gene exons; heavy solidlines, α gene introns; thin solid lines, pBR322 or pUC18 vector; andzig-zag lines, pUC18 polylinker regions.

FIG. 2: shows the basic expression vector, CLH3AXSV2[DHFR, ODC, or TPA],used for stable or transient transfections. The position of the XhoIsite used for insertion of α or β subunit sequences is shown. Doublesolid lines represent sequences needed for mammalian cell expression.The relative positions of the promoter, polyadenylation, and marker generegions are indicated. The single solid line represents thepBR322-derived pML-1 region necessary for propagation and selection (byampicillin resistance) in E. coli.

FIG. 3: part A shows the full-length TSHβ gene. The positions of thethree exons (I, II, and III), two introns (a, b), start ATG, and stopcodon (TAA) are indicated. The PvuII sites used to isolate the fragmentfrom which the partial constructions were derived are also indicated.Hatched boxes denote noncoding exon sequences. Part B shows the partialgenomic constructions used to compare TSHβ mRNA accumulation intransiently transfected COS-7 cells. TSHβ0.9 consists of the two codingexons separated by the endogenous IVS with all sequences upstream of thestart ATG and downstream of the TAA removed (sequence shown in Table 9).TSHβ1.2 and TSHβ2.0 contain, in addition, about 300 base pairs of intronA and were constructed by adding a synthetic splice donor (Δ) to allowsplicing of the truncated intron. TSHβ2.0 retained the endogenouspolyadenylation signal (Δ) and about 0.8 kb of additional 3' flankingsequence.

FIG. 4: describes CLH3AXSV2DHFR. This vector was constructed from thefollowing components: (i) the dihydrofolate reductase (DHFR)transcriptional unit (nucleotide numbers 1to 1925 of FIG. 4) whichconsists of the SV40 early region promoter (33, 34), the mouse DHFR gene(REFS) and the SV40 small T intron and early region polyadenylationsignal sequences (33, 34); (ii) the bacterial plasmid vector sequencesof pML (nucleotide numbers 2201 to 4542 of FIG. 4) derived from thepBR322 vector (29) from which a 1370 base pair sequence has been deleted(32); and (iii) the metallothionein promoter (nucleotide numbers 4542 to7514 of FIG. 4) derived from the mouse metallothionein-1 gene (30, 31)from which the introns and polyadenylation signal sequences have beenremoved; and (iv) the SV40 early region polyadenylation signal sequences(nucleotide numbers 7514 to 7751 of FIG. 4) (33, 34). The tPA analogueswere inserted into the vector as a SalI fragment at the unique XhoIsite. The orientation of the insert relative to the promoter andpolyadenylation sequences was determined by restriction enzyme analysis.

DETAILED DESCRIPTION AND BEST MODE Example 1 Plasmid constructions forthe expression of the common α and FSHβ subunits

A. The complete human α subunit genomic clone can be convenientlyobtained from a number of sources as a 17 kilobase pair (kb) EcoRIinsert in pBR322. Because the α promoter has been shown to be tissuespecific and would be unlikely to function efficiently in the tissueculture cells commonly used for heterologous gene expression, steps weretaken to remove all 5' flanking sequences. The presence of internalEcoRI sites necessitated several subcloning steps prior to assembly ofthe trimmed genomic sequence. The engineering strategy advantageouslyused is diagrammed in FIG. 1. Two pUC18 subclones were constructed, thefirst with the 8.0 kb BamHI-SacI 5' piece (pUCbs8.0), and the secondwith the 3' 2.7 kb SacI-EcoRI piece (pUCse2.7). To generate a terminuscompatible with the XhoI cloning sites in our expression vectors,pUCse2.7 was digested with EcoRI, the ends were blunted by treatmentwith the Klenow fragment of E. coli DNA polymerase I, and then SalIlinkers were attached in a ligation reaction. Subsequent SalIrestriction endonuclease digestion of the reaction mixture yielded a 2.7kb human α SalI piece in addition to the 2.6 kb vector fragment. The 2.7kb human α SalI piece was gel purified and re-inserted into into theSalI site of pUC18 (pUCss2.7). A clone was chosen which contained theSalI insert in the orientation which permitted isolation of the 2.7 kb αfragment as a SacI piece. This was gel purified and then inserted intothe SacI site of pUCbs8.0 to assemble the complete coding sequence ofthe human α gene as an 11 kb insert in pUC18. The insert could beexcised as a SalI fragment by virtue of a pre-existing SalI site in thepUC18 polylinker at the 5' end of the gene and the converted SalI site(from EcoRI) at the 3' end of the gene. The completed genomic αexpression construction, henceforth referred to as the "full-lengthgenomic α" sequence, included part of exon I (less the first 35nucleotides which comprise the 5' untranslated region of the mRNA), allof exons II, III, and IV as well as the intervening sequences, andapproximately two kilobase pairs (kb) of 3' flanking sequence. This wasinserted into the XhoI site of the CLH3AXSV2DHFR expression vector (FIG.2) so that transcription was directed by the mouse metallothionein-I(MT-I) promoter. The expression vector also contained the mousedihydrofolate reductase (DHFR) gene for a selectable and amplifiablemarker.

B. The human α subunit cDNA was engineered for expression by digestingthe full-length clone with NcoI, which spans the start ATG, and HindIII,which cleaves in the 3' untranslated region 215 base pairs (bp)downstream of the TAA stop codon. A 5' SalI site and Kozak consensussequence (27) was provided by synthetic oligonucleotides, and a 3' SalIsite by attaching linkers as described above. The DNA sequence of theengineered α subunit cDNA clone, which is approximately 600 bp inlength, is shown in Table 7. This was inserted into the XhoI site of theCLH3AXSV2DHFR expression vector (FIG. 2). The endogenous 5' untranslatedregion and 3' polyadenylation signal were removed from the cDNA clone inthe process of engineering and therefore were supplied by vectorsequences: the MT-I promoter and the simian virus 40 (SV40) earlypolyadenylation signal, respectively.

C. The human FSHβ partial genomic clone used in this study was a 2.0 kbDdeI-Sau3A segment which contained the protein coding region of exon IIin addition to 40 bp of sequence upstream of the start ATG, the proteincoding region of exon III, and the 1.6 kb intron which separates the twoexons (Table 7). The 5' DdeI and the 3' Sau3A sites had formerly beenconverted to EcoRI and BamHI sites, respectively, and therefore were notcompatible with the current expression vectors. The partial FSHβ genomicclone was therefore supplied with SalI termini by blunting as describedabove and attaching commercially prepared SalI linkers. The SalI piecewas then inserted into the XhoI site of CLH3AXSV20DC (FIG. 2), anexpression vector structurally similar to CLH3AXSV2DHFR except that theDHFR coding region was replaced with that for murine ornithinedecarboxylase (ODC).

Example 2 Comparison of the full-length genomic α cDNA α CLH3AXSV2DHFRtranscription units in stable transfections

The genomic α and cDNA α CLH3AXSV2DHFR constructions were compared bycotransfection of each α expression plasmid with the human FSHβCLH3AXSV20DC expression plasmid and measurement of FSH dimer production.

A. Twenty-four hours before the transfections were to be done, 100millimeter dishes were seeded with 7×10⁵ DUKX CHO cells (DHFR-minus).Calcium phosphate precipitations were obtained by adding 31 microliters(μl) of 2 molar (M) CaCl₂ to the plasmid DNA suspended in 500 μl oftransfection buffer [14 millimolar (mM) NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄,5.5 mM dextrose, and 20 mM HEPES (pH 7.5)]. Plasmid DNA amounts usedwere α (cDNA or genomic): 10 μg, and FSHβ: 30 μg. The precipitates wereallowed to form for 45 minutes at room temperature. The culture mediumwas removed from the cells and replaced with the DNA precipitate. Afterallowing the cells to sit at room temperature for 20 minutes, 5 ml ofculture medium was added to each dish and incubation was continued at37° C. for 4 hours. The cells were then shocked for 3.5 minutes in 15%glycerol at 37° C. After incubation for 48 hours at 37° C. in culturemedium the cells were split 1:10 and selection medium containing 0.02methotrexate (MTX) was added. The selection medium used was α-minusmodified Eagle's medium supplemented with 10% dialyzed fetal bovineserum and 1% L-glutamine. Ten to fourteen days later, foci were visibleand were transferred to 24-well plates. Culture media from these wereassayed for FSH dimer expression by using a specific monoclonal-basedradioimmunoassay (Serono Diagnostics, Randolph, Mass.). Positive cloneswere transferred to T-25 flasks in selection medium which contained anincreased MTX concentration of 0.1 μM. When the cultures reachedconfluence the media were again assayed for FSH dimer and the cells werecounted to calculate the picogram per cell per 24 hour expressionlevels.

B. Human FSH dimer secretion levels measured in seven randomly selectedclones from each of the human α genomic/FSHβ and human α cDNA/FSHβcontransfections are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        α GENOMIC-DHFR/FSHβ-ODC                                                              α cDNA-DHFR/FSHβ-ODC                             GFSH     pg/cell/24h in                                                                             CFSH       pg/cell/24h in                               Cell Line                                                                              0.1 μM MTX                                                                              Cell Line  0.1 μM MTX                                ______________________________________                                        1        0.43         18        0.003                                         3        1.95         37        0.03                                          4        0.50         51        0.08                                          5        0.70         57        0.02                                          7        1.44         60        0.056                                         8        1.18         66        0.051                                         9        2.56         70        0.013                                         Avg.     1.25         Avg.      0.04                                          ______________________________________                                    

The results show that FSH dimer expression is greatly enhanced in cellstransfected with the full-length genomic α subunit sequence. Theaveraged expression levels indicate that the surprisingly largemagnitude of the enhancement seen in this particular experiment wasapproximately thirty-fold.

C. To further demonstrate the superiority of the full-genomic αsequence, stable cell lines were transfected with the CLH3AXSV2DHFRexpression vector that contained either the human α cDNA or the human αgenomic clone. Expression rates of the free α subunit were compared. Inall cases, the expression of human α subunit, as determined by asensitive and specific radioimmunoassay, was never greater than 0.05pg/cell/24h for the cDNA-containing cell lines. As detailed in Table 2,cells that were transfected with the genomic α clone expressed 5-20times greater levels of the protein:

                  TABLE 2                                                         ______________________________________                                        Alpha        Expression Level                                                 Cell Line    pg/cell/24h                                                      ______________________________________                                         2           0.32                                                             10           0.36                                                             12           0.57                                                             17           0.37                                                             18           0.39                                                             38           1.28                                                             47           0.63                                                             51           0.26                                                             ______________________________________                                    

Example 3 Thyroid stimulating hormone constructions

To demonstrate the effectiveness and broad application of thisinvention, stably transfected CHO cell lines were prepared bycotransfection of the full-length human genomic α sequence inCLH3AXSV2DHFR with a partial genomic TSHβ sequence in CLH3AXSV2ODC. Thepartial genomic TSBβ fragment used in this experiment consisted of theprotein coding regions of exon II and III, and the 0.5 kb intron whichseparated the two exons (Table 9). All 5' and 3' regions flanking theprotein coding sequence of TSHβ were removed in this particularconstruction. Following cotransfection with the two expression vectors,stable cell lines were cultured and analyzed for their ability toexpress TSH. Expression levels of the dimer, as determined by asensitive and specific radioimmunoassay, are listed in Table 3. Previousstudies with the transfection of the cDNA for the α subunit with thegenomic TSHβ clone had demonstrated that expression levels were usuallybelow the sensitivity of the radioimmunoassay, usually less than 0.02pg/cell/24 h.

                  TABLE 3                                                         ______________________________________                                        TSH          Expression Rate                                                  Cell Line    pg/cell/24h                                                      ______________________________________                                         7           0.19                                                              8           0.22                                                             10           0.24                                                             12           0.24                                                             37           0.13                                                             48           2.97                                                             ______________________________________                                    

TSH production, then, like FSH production, can be greatly enhanced byuse of the full genomic α sequence, rather than the α cDNA sequence, inmammalian cell transfections. In this experiment the range of TSHproduction enhancement was 6 to 100 fold.

Example 4 Introns and expression enhancement

The human α genomic construction differed from the human α cDNAconstruction not only in that it contained introns, but also in that itcontained endongenous 5' untranslated sequence, the endogenouspolyadenylation signal, and additional 3' flanking sequences. Therefore,one could not infer from the results of the previous set experimentswhich genomic regions contributed to the enhanced expression.

A. To determine if the introns within the genomic α sequence wereresponsible for the elevated α subunit levels, we inserted a 2 kbXbaI-PstI portion of the human α intron A between the mouse MT-I 5'untranslated region and the α cDNA sequence in the CLH3AXSV2TPA vector(FIG. 2). The truncated intron retained the endogenous splice acceptor,but the splice donor was supplied by a synthetic oligonucleotide.

B. Another plasmid was constructed to test the effect of a heterologousintron on α subunit expression. In this construction, a 130 bp intronfrom the MOPC41 immunoglobulin κ gene (5) was inserted between the mouseMT-I 5' untranslated region and the α cDNA sequence. No transcriptionalenhancer elements were included in this particular intervening sequence.

C. The intron-containing α cDNA constructs were compared to the originalα cDNA construct and the full length genomic α sequence by transienttransfection of COS-7 cells with the plasmid DNA and analysis of mRNAlevels by northern blotting. In this experiment, the tissue plasminogenactivator (tPA) cDNA served as an internal standard and was used tocorrect for the variations in the transfection efficiency of differentplasmid constructions and thus normalize measured α subunit mRNA levels.Transfections were done in duplicate using the DEAE-dextran protocol ofSeed and Aruffo (28). Two days after transfection, total cellular RNAwas isolated from the cells. The RNA (5 micrograms) was fractionated onformaldehyde gels and then transferred to nylon membranes using standardnorthern blotting techniques. The membranes were then hybridized toeither a ³² P-labeled human α or a ³² P-labeled tPA probe and theresulting signals were quantitated on a Betascope Model 603 blotanalyzer (Betagen Corp., Waltham, Mass.). Normalized α mRNA values forrelative comparisons were calculated by dividing the number of α countsby the number of tPA counts and then averaging the numbers obtained forduplicate samples. Results of the experiment are shown in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    Plasmid      Betascope counts                                                                         Normalized Values                                                                       Average of                                  Construction α probe                                                                      tPA probe                                                                           (α/tPA)                                                                           Duplicates                                  __________________________________________________________________________    1. α genomic                                                                       A  6315                                                                               958  6.6        7                                                     B  7836                                                                              1177  6.7                                                   2. α cDNA + α IVS                                                            A 25353                                                                              1294  19.6      21                                                     B 34616                                                                              1559  22.2                                                  3. α cDNA + Ig IVS                                                                 A 31743                                                                              1690  18.8      17                                                     B 37495                                                                              2327  16.1                                                  4. α cDNA                                                                          A  3891                                                                              2608  1.5        1                                                     B  3671                                                                              3341  1.1                                                   __________________________________________________________________________

The results indicated that the cDNA constructions which contained eitherthe human α intron (No. 2) or the immunoglobulin intron (No. 3) weremore efficient as the full length genomic sequence (No. 1) in theaccumulation of α subunit mRNA. Specifically, the normalized α subunitmRNA levels were 7- to 21-fold higher than those produced by the cDNAconstruct (No. 4) without an intron. We have therefore unexpectedlydiscovered that introns, and not the 5' or 3' regions which flank the αsubunit protein coding sequence, are primarily responsible for theincreased expression levels and that the effect is due, at least inpart, to an increased accumulation of α subunit mRNA.

Example 5 Expression enhancement not due to increased transcriptionrates

Nuclear runoff transcription assays were used to determine if the highlevel of genomic α-induced mRNA accumulation was due to an increasedtranscription rate. Transient transfection of COS-7 cells with the αgenomic and α cDNA-containing CLH3AXSV2TPA plasmids was done asdescribed in Example 4. Standard methods were used in the preparation ofnuclei and for the runoff transcription assay (Current Protocols inMolecular Biology; Ausubel, F.M. et al., ed.; John Wiley & Sons, NewYork, N.Y.). The DNA probes for α, DHFR, and tPA were gel purifiedinsert sequences and approximately 0.25 μg of each were slot blotted induplicate onto nitrocellulose membranes. The membranes were hybridizedto [³² P-UTP] labeled nuclear runoff RNA prepared from COS-7 cells thathad been transfected with no DNA (mock), the full-length human α genomicclone in CLH3AXSV2TPA, or the human α cDNA clone in CLH3AXSV2TPA. Thehybridization signal was quantitated on a Betascope Model 603 blotanalyzer (Betagen, Corp., Waltham, Mass.). Normalized values for thetranscription rate were obtained by dividing the averaged α counts bythe averaged tPA counts. The monkey DHFR nuclear runoff RNA should nothybridize to the mouse DHFR DNA probe sequence in the conditions usedfor this experiment, and therefore serves as a negative control. Therelative transcription rate results are summarized in Table 5, below.

                  TABLE 5                                                         ______________________________________                                               Nuclear runoff RNA                                                                                  αgenomic/                                         Mock COS-7                                                                             α cDNASV2TPA                                                                         SV2TPA                                           DNA Probes                                                                             1       2      1     2      1     2                                  ______________________________________                                        α cDNA                                                                           119     160    2093   2155   492  620                                tPA      387     413    18022 17667  8486  7388                               DHFR     230     307     54     0     32    765*                              Rate Ratios:                                                                           --         0.12         0.07                                         α/tPA                                                                   ______________________________________                                         *Non-specific background rendered this value artifically high            

In this particular experiment the runoff transcription rate of the αcDNA (0.12) was surprisingly higher than that of the genomic α sequence(0.07). An increased transcription rate is therefore not the mechanismby which the genomic α sequence generates higher α subunit expressionlevels.

Example 6 TSHβ Subunit expression is intron-dependent

A. The full-length genomic TSHβ gene (14a) is diagrammed in FIG. 3, PartA. The positions of the three exons (I, II, and III), two introns (a,b), start ATG, and stop codon (TAA) are indicated. The PvuII sites wereseparated by approximately 2 kb of DNA sequence which included exons IIand III and contained the complete protein coding sequence for TSHβ. TheTSHβ partial genomic constructions used in this study were derived fromthe 2 kb PvuII fragment and are diagrammed in Table 3B. TSHβ 0.9 (0.9kb) was the same construct used in stable transfections in Example 1 andconsisted of the two coding exons separated by the endogenousintervening sequence with all sequences upstream of the start ATG anddownstream of the TAA removed. TSHβ 1.2 (1.2 kb) and TSHβ 2.0 (2.0 kb)contained, in addition, about 300 base pairs of intron A and wereconstructed by adding a synthetic splice donor to allow splicing of thetruncated intron. TSHβ 2.0 retained the endogenous polyadenylationsignal and about 0.8 kb of additional 3' flanking sequence.

B. Duplicate cultures (A, B) of COS-7 cells were transfected, using theprotocol described in Example 4, with CLH3AXSV2DHFR plasmids whichcontained one of the following partial genomic fragments: (1) TSHβ 0.9,(2) TSHβ 1.2, or (3) TSHβ 2.0. The genes were inserted into the XhoIcloning site so that transcription would be initiated by the MT-Ipromoter and, in the case of TSHβ 0.9 and TSHβ 1.2, terminated by theSV40 early polyadenylation signal. After 48 hours of incubation, totalcellular RNA was isolated from the cells. The RNA (9 micrograms) wasfractionated on formaldehyde gels and then transferred to nylonmembranes using standard northern blotting techniques. The membraneswere hybridized to either a ³² P-labeled mouse MT-I probe (to detect theTSHβ mRNA which also contains about 50 base pairs MT-I 5' untranslatedsequence) or a ³² P-labeled DHFR probe (to compare transfectionefficiencies). The resulting signals were quantitated on a BetascopeModel 603 blot analyzer (Betagen Corp., Waltham, Mass.). Normalized TSHβmRNA values for relative comparisons were calculated by dividing MT-Icounts by DHFR counts. Normalized values obtained for duplicatetransfections were then averaged and divided by the number obtained forTSHβ 0.9. The comparative results obtained for accumulation of mRNA aresummarized in Table 6.

                  TABLE 6                                                         ______________________________________                                                 Betascope Counts                                                                         Normalized Relative                                       Plasmid        MT      DHFR   Values   Values                                 Construction   Probe   Probe  (MT/DHFR)                                                                              (averaged)                             ______________________________________                                        1. TSHβ 0.9                                                                       A      983    3985   0.25     1                                               B      834    4118   0.20                                            2. TSHβ 1.2                                                                       A     4480     254   17.6     60                                              B     2748     285   9.6                                             3. TSHβ 2.0                                                                       A     1381    1209   1.2      5                                               B     1632    1254   1.3                                             ______________________________________                                    

C. The results indicated that the two constructs which retained aportion of the first intron (TSHβ 1.2 and TSHβ 2.0) yielded 5-60 foldhigher mRNA accumulation than did the construct which contained nosequences from the first intron. Therefore TSHβ gene expression, like αsubunit gene expression, is intron-dependent. Of interest was theunexpected observation that intron B, which was present in all theconstructs and was in the protein coding region, did not confer optimalenhancement. This suggested either that there are specific sequences inthe first intron of the TSHβ gene which increased mRNA accumulation or,more likely, that the position of the intron (close to the 5' end of themRNA) was the crucial factor. That intron-dependent gene expression maybe affected by the placement of the intron in the transcription unit issupported by preliminary experiments with the human α cDNA. Insertion ofthe κ immunoglobulin gene intron in the 3' untranslated region of themRNA, between the α cDNA and the SV40 polyadenylation region, resultedin no increased mRNA accumulation when compared to the unaltered α cDNA.

D. Since TSHβ 1.2 yielded 10-fold higher amounts of mRNA than TSHβ 2.0,it is probable that the endogenous polyadenylation signal and 3'flanking sequence carried on TSHβ 2.0 are not required for efficientmRNA formation, and may actually be detrimental. Alternatively, thedisparity in mRNA accumulation between TSHβ 1.2 and TSHβ 2.0 may berelated to the different distances between the MT-I promoter and theSV40 early promoter (driving the DHFR) in the two plasmid constructions.The closer proximity (0.8 kb) of the two promoters in the TSHβ 1.2construction may permit increased interaction between the SV40 enhancerelements and the MT-I promoter, thereby increasing the rate oftranscription from the latter.

Because of the similarity of α and TSHβ gene structure among variousmammals, we expect that our findings based on the human α and TSHβsubunits apply equally to those of other species, such as the bovine,equine, porcine, baboon and monkey. Likewise, LHβ and FSHβ subunit genesmay show a similar dependence on genomic DNA structure for optimalexpression and these requirements should apply across species.

Construction of expression vectors will be advantageously simplified byapplication of the knowledge that certain genomic regions, such asintrons, are important for mRNA accumulation, and that the position ofthese regions in the transcription unit may be important. For example,these genomic regions may be subcloned or synthesized and included inconstructions with the α and β subunit cDNA sequences. The cDNA clonesare often easier to obtain and to engineer than the much larger genomicclones. Thus, this discovery will simplify the development of cell lineswhich produce high levels of hormone and, ultimately, will permit largescale production of these proteins at a lower cost. These and otheraspects and variations of the present invention will now be readilyapparent to those skilled in the art and are not deemed to depart fromeither the spirit or the scope of the present invention.

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Except as otherwise stated, procedures and techniques used are generallyas reported and conventional in the art such as is described by Maniatiset al, A Cloning Manual, Cold Spring Harbor (1982). All references citedabove or in the specification are fully incorporated herein byreference.

                                      TABLE 7                                     __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                     421AGTTGTTTAGTGTTTATGG CTTTGTGAGATAAAACTCTCCTTTT CCTTACCATACCACTT480          481TGACACGCTTCAAGGATATA CTGCAGCTTTACTGCCTTCCTCCTTA TCCTACAGTACAAT540           ##STR8##                                                                     __________________________________________________________________________

The engineered alpha subunit cDNA sequence. Restriction endonucleasesites and Kozak consensus region which were supplied by syntheticoligonucleotides are indicated. The alpha subunit amino acid sequence isalso shown.

                                      TABLE 8                                     __________________________________________________________________________     ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                    1675ATTTCTGTCTCATTTTGAC TAAGCTAAATAGGAACTTCCA CAATACCATAACCTAACTCT1734         ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                    __________________________________________________________________________

The engineered human FSH beta subunit partial genomic sequence.Positions of the terminal SalI sites are indicated. Nucleotides in lowercase at the 3' terminus results from attachment of synthetic linkers.The FSH beta amino acid sequence marks the coding regions of exons IIand III. The dotted line represents uncharacterized intervening sequence(IVS).

                                      TABLE 9                                     __________________________________________________________________________     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                    181TGTAGTTCATGTCACTTCTTTTGGCTGTA AATTATATAAGCCCTGAAGAAGTCCATTCCT240           241ATATAGAAAGGAAATGAAATAAATCACAA CCTCATTTCCCAAATCTAATGGTTATTGGCT300           301CCTTAGAAGCAGAGTACACAGGTTACAAT ATTATGTGAATCTACTCAGCACAATGGATAC360           361GCATAATTTTATAACAGTTTTGTGTCCCA GCTTTACTTAAACCTTATCTTGTTCCCATGA420           421TCAACGATGAAAGAGAGGAGGGTCTCACT TTTGTCTCTGTAGAATTCAACGTGGTTAAGT480           481TGGTATTGGAGAATGGGGCTAAGCAATTC TTTCCCAGTTGTATTTGTGATGAAGGAATAT540           541AAGTGAATTTATTTTTATGTTTCTATTAT CTATATGTTTCCTAAAGTCCTGTCACATTAT600            ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                    __________________________________________________________________________

The engineered TSH beta subunit partial genomic sequence. SalI sites andKozak consensus sequence supplied by synthetic oligonucleotides areindicated. The TSH beta amino acid sequence marks the coding regions ofexons II and III.

What is claimed is:
 1. A method for the production of thyroidstimulating hormone comprising the steps of:a) providing a first vectorcomprising a promoter, a DNA fragment encoding for the alpha subunit ofsaid thyroid stimulating hormone and comprising at least one intron, anda terminating sequence, and a second vector comprising a promoter, a DNAfragment TSHB 1.2 or TSHB 2.0 encoding for the beta subunit of saidthyroid stimulating hormone consisting essentially of coding exons IIand III separated by an endogenous intervening sequence, and one intronabout 300 base pairs in length positioned upstream of and adjacent toexon II, and a terminating sequence; b) transforming host cells withsaid vectors; and c) culturing said transformed cells under conditionswhereby said thyroid stimulating hormone is produced.
 2. The method ofclaim 1 wherein said DNA fragment encoding said α subunit furthercomprises a plurality of introns, 3' and 5' flanking regions, endogenous5' untranslated sequence and polyadenylation signal.
 3. The method ofclaim 1 wherein said selected dimeric protein is human thyroidstimulating hormone.
 4. The method of claim 1 wherein said selecteddimeric protein is non-human thyroid stimulating hormone.
 5. The methodof claim 3 wherein the DNA fragment for the beta subunit of humanthyroid stimulating hormone is a 1.2 kb DNA fragment containing the twocoding exons separated by an endogenous intervening sequence, butwithout the endogenous polyadenylation signal or additional 3' flankingsequence.
 6. The method of claim 3 wherein the DNA fragment for the betasubunit of human thyroid stimulating hormone is a 2.0 kb DNA fragmentcontaining the two coding exons separated by an endogenous interveningsequence, and including the endogenous polyadenylation signal and 0.8 kbof additional 3' flanking sequence.